We have proposed that the entry into the memory B cell compartment is regulated by BCR specificity for foreign antigen, rather than affinity per se. We suggest that specificity-based selection takes place in an iterative fashion via positive selection for affinity for foreign antigen and negative selection against affinity for self antigens in conjunction with V region hypermutation in the germinal center (GC). We use the term "specificity maturation" to describe this process. Since many of the BCRs expressed by primary peripheral B cells in mice and men display "multireactivity" for foreign and autoantigens, defects or perturbations in specificity maturation are predicted to directly contribute to the development of systemic autoimmune diseases such as SLE through enhanced entry of autoreactive clonotypes into the memory B cell pool. In the previous funding period we conducted tests of the specificity maturation hypothesis using a novel Ig transgenic system. A transgenic H chain was engineered to contain an arginine (R) in the V region that dramatically increases affinity for DMA-based autoantigens, but does not alter affinity for the foreign antigen arsonate (Ars) of the BCR expressed by a particular B cell clonotype (termed "canonical"). Canonical B cells with and without the R55 mutation both populate the mature, follicular compartment in similar numbers. However, mature R55 canonical clonotypes express low levels of BCR, due to autoantigen engagement. Nonetheless, such B cells do not display conventional features of anergy in vitro, and they can mount a robust early Ars-KLH-driven proliferative response in vivo. However, these clonotypes enter, but do not appear to expand in GCs, and inefficiently populate the anti-Ars memory compartment. These data support the general tenets of the specificity maturation hypothesis by suggesting that multireactive clonotypes expressing BCRs with potentially pathological levels of autoreactivity are eliminated during foreign antigen-driven GC responses. However, the limited resolution of the experiments conducted so far have not allowed detailed mechanistic insight into how this tolerance pathway operates, and how it may fail in autoimmunity. As such, in this competitive renewal we propose to define the relative roles of pre-GC, GC and post-GC tolerance checkpoints in the regulation of the activity of canonical R55 clonotypes, and determine how genetic defects previous shown to contribute to the breakdown of B cell tolerance influence the action of these checkpoints on this clonotype. Systemic autoimmune diseases such as Lupus often cause debilitating illness and are a major health issue in this country, particularly among women and minority groups. There are currently no cures for these diseases and available therapies are complex. More complete knowledge of the root causes of these diseases is required to improve this situation. Acquiring this knowledge is the goal of the experiments in this proposal. [unreadable] [unreadable] [unreadable]